Soluble interleukin-7 receptor (sIL7R) modulating therapy to treat autoimmune diseases and cancer

ABSTRACT

The present invention includes compositions and methods for treating an autoimmune disorder or a cancer in a subject in need thereof, the method comprising: administering an effective amount of a composition comprising an oligonucleotide that specifically binds a complementary sequence of the Interleukin-7 receptor (IL7R) pre-mRNA that influences splicing of exon 6, wherein the SM-ASO increases or decreases inclusion of exon 6 in IL7R pre-mRNAs and respectively decreases or increases expression of the soluble isoform of IL7R (sIL7R). In certain embodiments, the oligonucleotide is an antisense oligonucleotide (ASO), or a splice-modulating antisense oligonucleotide (SM-ASO).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to PCT/US2019/023719, filed Mar. 22,2019 and U.S. Provisional Application Ser. No. 62/646,716, filed Mar.22, 2018, the entire contents of which are incorporated herein byreference.

STATEMENT OF FEDERALLY FUNDED RESEARCH

This invention was made with government support under F32-NS087899awarded by NIH. The government has certain rights in the invention.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to the field of novel therapiesthat reduce or increase soluble IL7R (sIL7R) to treat autoimmunediseases (e.g., multiple sclerosis) or cancer, respectively.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with multiple sclerosis, as an example.

Multiple Sclerosis (MS) is a chronic autoimmune disease characterized byself-reactive immune cell-mediated damage to neuronal myelin sheaths inthe central nervous system (CNS) that leads to axonal demyelination,neuronal death and progressive neurological dysfunction. Up to date,there is no cure for the disease and available treatments can only slowdown disease progression, often by globally suppressing the immunesystem, causing a plethora of adverse side effects that could be severeor lethal. This global immunosuppression is the major limitation ofcurrent therapies.

The breach of immunological tolerance that leads to MS is thought tooriginate from complex interactions between environmental and geneticfactors. Under this view, the genetic background of an individual couldgenerate an environment permissive for the survival of self-reactivelymphocytes, which could be subsequently activated by the presence of anenvironmental trigger, usually in the form of viral or bacterialinfection.

The present inventors and others have previously shown that the variantrs6897932 (C/T, where C is the risk allele) within exon 6 of theInterleukin-7 receptor (IL7R) gene is strongly associated with increasedMS risk (Gregory et al., 2007; International Multiple Sclerosis Geneticset al., 2007; Lundmark et al., 2007). Furthermore, the present inventorsshowed that the risk ‘C’ allele of this variant increases skipping ofthe exon (Evsyukova et al., 2013; Gregory et al., 2007), leading toup-regulation of sIL7R (Hoe et al., 2010; Lundstrom et al., 2013).Importantly, sIL7R has been shown to exacerbate the clinical progressionand severity of the disease in the Experimental AutoimmuneEncephalomyelitis (EAE) mouse model of MS, presumably by potentiatingthe bioavailability and/or bioactivity of IL7 cytokine (Lundstrom etal., 2013). Further supporting a role of sIL7R in the pathogenesis ofmultiple sclerosis, and perhaps autoimmunity in general, elevated levelsof sIL7R protein or RNA have been reported in patients of multiplesclerosis (McKay et al. 2008), rheumatoid arthritis (Badot et al.,2011), type 1 diabetes (Monti et al., 2013), and systemic lupuserythematosus (Lauwerys et al., 2014). Collectively, these data linkelevated levels of sIL7R to the pathogenesis of MS and autoimmunity, andposition alternative splicing of IL7R exon 6 as a novel therapeutictarget for MS and autoimmunity.

While these references teach that the presence of sIL7R correlates withmultiple sclerosis, and animal studies demonstrate that sIL7Rexacerbates an MS-like condition, a need remains for novel compositionand methods for treating autoimmune diseases, such as MS, by targetingthe production of sIL7R.

Many different etiologies exist for autoimmune diseases, and each ofthese can be the target of accurate or personalized therapies. In thisinvention, the present inventors address one such etiology caused byelevated levels of sIL7R, which is likely to affect as many as 60% of MSpatients and a significant number of patients with other autoimmunedisorders caused by elevated sIL7R.

Immuno-oncology is a rapidly growing field that holds great promise forpatients with heretofore intractable cancers; however, the impact ofimmunotherapy has been limited by very low response rates in somecancers and individuals.

Antisense oligonucleotide therapy targets a genetic sequence of aparticular gene that is causative of a particular disease with a shortoligonucleotide that is complementary to a target sequence. Typically, astrand of nucleic acids is designed (DNA, RNA or a chemical analogue)that binds to the messenger RNA (mRNA) or pre-mRNA of the targetsequence. In the case of splice-modulating antisense oligonucleotides(SM-ASOs), the complementary nucleic acid is designed to bind a specificsequence in a pre-mRNA that modifies the exon content of the resultingmRNA. Antisense oligonucleotides have been used to target diseases suchas cancers, diabetes, amyotrophic lateral sclerosis (ALS), Duchennemuscular dystrophy, spinal muscular atrophy, Ataxia-telangiectasia,asthma, and arthritis. Several antisense oligonucleotide drugs have beenapproved by the U.S. Food and Drug Administration (FDA), for thetreatment for cytomegalovirus retinitis, homozygous familialhypercholesterolemia, Duchenne muscular dystrophy, and spinal muscularatrophy, to name a few, with the latter two been SM-ASOs. However, ineach case the oligonucleotide target sequence must be tailored to thespecific etiology underlying the disease in question.

SUMMARY OF THE INVENTION

In one embodiment, the present invention includes a method of treating adisease or condition with elevated levels of a soluble isoform ofInterleukin 7 receptor (sIL7R) in a subject in need thereof, the methodcomprising: administering an effective amount of a compositioncomprising an oligonucleotide that specifically binds to a sequence ofthe Interleukin-7 receptor (IL7R) pre-mRNA that influences splicing ofexon 6, wherein the oligonucleotide increases inclusion of exon 6 inIL7R pre-mRNAs and decreases expression of the soluble isoform of IL7R(sIL7R). In one aspect, the oligonucleotide is an antisenseoligonucleotide (ASO) or a splice-modulating antisense oligonucleotide(SM-ASO). In another aspect, the oligonucleotide in the compositionspecifically binds to a sequence in IL7R pre-mRNA in at least one of thegroup consisting of an exonic splicing silencer (ESS) and/or an intronicsplicing silencer (ISS), thereby enhancing inclusion of exon 6 in IL7Rpre-mRNAs, and reducing expression of sIL7R. In another aspect, theoligonucleotide in the composition specifically binds to a sequence onIL7R pre-mRNA at intron-exon splice sites, branchpoint sequences, and/orpolypyrimidine tracts. In another aspect, at least one or morenucleotide(s) in the oligonucleotide contains a non-naturally occurringmodification comprising modifications or substitutions of: (1) theribose or other sugar units, (2) bases, or (3) the backbone, selectedfrom: one or more phosphorothioate, phosphorodithioate, phosphodiester,methyl phosphonate, phosphoramidate, methylphosphonate, phosphotriester,phosphoroaridate, morpholino, amidate carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and/or alkylsilyl substitutions, partially or completelymodified backbones, such as fully modified sugar phosphate backbone, alocked nucleic acid backbone, a peptidic backbone, a phosphotriesterbackbone, a phosphoramidate backbone, a siloxane backbone, acarboxymethylester backbone, an acetamidate backbone, a carbamatebackbone, a thioether backbone, a bridged methylene phosphonatebackbone, a phosphorothioate backbone, a methylphosphonate backbone, analkylphosphonate backbone, a phosphate ester backbone, analkylphosphonothioate backbone, a phosphorodithioate backbone, acarbonate backbone, a phosphate triester backbone, a carboxymethyl esterbackbone, a methylphosphorothioate backbone, a phosphorodithioatebackbone, a backbone having p-ethoxy linkages, sugar modifications suchas 2′-O-methyl (2′-O-methylnucleotides), 2′-O-methyloxyethoxy(2′-O-MOE), a 2′-O-alkyl modified sugar moiety, or a bicyclic sugarmoiety, nucleotide mimetics, peptide nucleic acids (PNA), morpholinonucleic acids, cyclohexenyl nucleic acids, anhydrohexitol nucleic acids,glycol nucleic acid, threose nucleic acid, and locked nucleic acids(LNA), and combinations of two or more of any of the foregoing. Inanother aspect, at least one or more nucleotide(s) in theoligonucleotide contains a non-naturally occurring modification to thenucleotide bases. In another aspect, the oligonucleotide is selectedfrom any of the SEQ IDs in Table 1 (SEQ ID NOS:1-13), or portionsthereof, either alone or in combination, or a sequence having at least70, 75, 80, 84, 85, 88, 92, 93, 94, 95, or 96% identity over the fulltarget sequences within IL7R RNAs. In another aspect, theoligonucleotide targets any of the SEQ IDs in Table 1 (SEQ ID NOS:1-13),either fully or partially. In another aspect, the composition furthercomprises a pharmaceutically acceptable excipient, salts, or carrier. Inanother aspect, the disease or condition is an autoimmune disorder isselected from at least one of the following: multiple sclerosis, type Idiabetes, rheumatoid arthritis, systemic lupus erythematosus, atopicdermatitis, ankylosing spondylitis, primary biliary cirrhosis, orinflammatory bowel syndromes such as ulcerative colitis, Crohn's diseaseor any other conditions where sIL7R is elevated when compared to anormal subject without a disease or condition. In another aspect, thedisease or condition is an inflammatory disease or condition. In anotheraspect, the oligonucleotide enhances the degradation of IL7R mRNAs thatlack exon 6 by targeting an IL7R exon 5-exon 7 boundary, e.g., withASOs, siRNAs, shRNAs that decrease stability of sIL7R RNA (e.g.,increase degradation), and/or ASOs that decrease translation of sIL7RRNA. In another aspect, the method further comprises a combinationtherapy of the SM-ASO and one or more active agents effective fortreating autoimmune diseases such as, but not limited to, mitoxatrone,interferon beta-1a, PEG- interferon beta-1a, azathioprine, fingolimod,natalizumab, methylprednisolone, or ocrelizumab. In another aspect, themethod further comprises steps of obtaining cells from the patient andmodifying the cells to transiently or permanently express theoligonucleotide that specifically binds to a sequence of theInterleukin-7 receptor (IL7R) pre-mRNA that influences splicing of exon6. In another aspect, the method further comprises generating a vectorthat expresses the oligonucleotide that specifically binds to a sequenceof the Interleukin-7 receptor (IL7R) pre-mRNA that influences splicingof exon 6 for use in gene therapy, and treating the patient with thevector.

In another embodiment, the present invention includes a compositioncomprising an oligonucleotide that is an antisense oligonucleotide (ASO)or a splice-modulating antisense oligonucleotide (SM-ASO), thatspecifically binds to a sequence in pre-mRNAs of Interleukin 7 receptor(IL7R) that influences splicing of exon 6, wherein the SM-ASO increasesinclusion of exon 6 in IL7R pre-mRNAs and decreases expression of thesoluble isoform of IL7R (sIL7R). In one aspect, the oligonucleotide inthe composition specifically binds to a sequence in IL7R pre-mRNA in atleast one of the group consisting of an exonic splicing silencer (ESS)and/or an intronic splicing silencer (ISS), thereby enhancing inclusionof exon 6 in IL7R pre-mRNAs, and reducing expression of sIL7R. Inanother aspect, the oligonucleotide in the composition specificallybinds to a sequence on IL7R pre-mRNA at intron-exon splice sites,branchpoint sequences, and/or polypyrimidine tracts. In another aspect,at least one or more nucleotide(s) in the oligonucleotide contains anon-naturally occurring modification comprising modifications orsubstitutions of: (1) the ribose or other sugar units, (2) bases, or (3)the backbone, selected from: one or more phosphorothioate,phosphorodithioate, phosphodiester, methyl phosphonate, phosphoramidate,methylphosphonate, phosphotriester, phosphoroaridate, morpholino,amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate,sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilylsubstitutions, partially or completely modified backbones, such as fullymodified sugar phosphate backbone, a locked nucleic acid backbone, apeptidic backbone, a phosphotriester backbone, a phosphoramidatebackbone, a siloxane backbone, a carboxymethylester backbone, anacetamidate backbone, a carbamate backbone, a thioether backbone, abridged methylene phosphonate backbone, a phosphorothioate backbone, amethylphosphonate backbone, an alkylphosphonate backbone, a phosphateester backbone, an alkylphosphonothioate backbone, a phosphorodithioatebackbone, a carbonate backbone, a phosphate triester backbone, acarboxymethyl ester backbone, a methylphosphorothioate backbone, aphosphorodithioate backbone, a backbone having p-ethoxy linkages, sugarmodifications such as 2′-O-methyl (2′-O-methylnucleotides),2′-O-methyloxyethoxy (2′-O-MOE), a 2′-O-alkyl modified sugar moiety, ora bicyclic sugar moiety, nucleotide mimetics, peptide nucleic acids(PNA), morpholino nucleic acids, cyclohexenyl nucleic acids,anhydrohexitol nucleic acids, glycol nucleic acid, threose nucleic acid,and locked nucleic acids (LNA), and any combinations of two or more ofany of the foregoing. In another aspect, at least one or morenucleotide(s) in the oligonucleotide contains a non-naturally occurringmodification to the nucleotide bases. In another aspect, theoligonucleotide is selected from any of the SEQ IDs in Table 1 (SEQ IDNOS:1-13), or portions thereof, either alone or in combination, or asequence having at least 70, 75, 80, 84, 85, 88, 92, 93, 94, 95, or 96%identity over the full target sequence within IL7R RNAs. In anotheraspect, the oligonucleotide targets any of the SEQ IDs in Table 1 (SEQID NOS:1-13), either fully or partially. In another aspect, thecomposition further comprises a pharmaceutically acceptable excipient,salts, or carrier. In another aspect, the composition is adapted foradministration to treat an autoimmune disorder selected from at leastone of the following: multiple sclerosis, type I diabetes, rheumatoidarthritis, systemic lupus erythematosus, atopic dermatitis, ankylosingspondylitis, primary biliary cirrhosis, inflammatory bowel syndromessuch as ulcerative colitis and Crohn's disease, or any other conditionswhere sIL7R is elevated. In another aspect, the oligonucleotide enhancesthe degradation of IL7R mRNAs that lack exon 6 by targeting an IL7R exon5-exon 7 boundary, e.g., with ASOs, siRNAs, shRNAs that decreasestability of sIL7R RNA (e.g., increase degradation), and/or ASOs thatdecrease translation of sIL7R RNA.

In yet another embodiment, the present invention includes a method ofincreasing inclusion of exon 6 of an Interleukin-7 receptor (IL7R)pre-mRNA, the method comprising: contacting a splice modulatingantisense oligonucleotide (SM-ASO) that specifically binds to a sequenceof the Interleukin-7 receptor (IL7R) pre-mRNA that influences splicingof exon 6, wherein the SM-ASO increases inclusion of exon 6 in IL7Rpre-mRNAs and decreases expression of the soluble isoform of IL7R(sIL7R). In one aspect, the SM-ASO in the composition specifically bindsto a sequence in IL7R pre-mRNA in at least one of the group consistingof an exonic splicing silencer (ESS) and/or an intronic splicingsilencer (ISS), thereby enhancing inclusion of exon 6 in IL7R pre-mRNAs,and reducing expression of sIL7R. In another aspect, the SM-ASO in thecomposition specifically binds to a sequence on IL7R pre-mRNA atintron-exon splice sites, branchpoint sequences, and/or polypyrimidinetracts. In another aspect, at least one or more nucleotide(s) in theSM-ASO contains a non-naturally occurring modification comprisingmodifications or substitutions of: (1) the ribose or other sugar units,(2) bases, or (3) the backbone, selected from: one or morephosphorothioate, phosphorodithioate, phosphodiester, methylphosphonate, phosphoramidate, methylphosphonate, phosphotriester,phosphoroaridate, morpholino, amidate carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and/or alkylsilyl substitutions, partially or completelymodified backbones, such as fully modified sugar phosphate backbone, alocked nucleic acid backbone, a peptidic backbone, a phosphotriesterbackbone, a phosphoramidate backbone, a siloxane backbone, acarboxymethylester backbone, an acetamidate backbone, a carbamatebackbone, a thioether backbone, a bridged methylene phosphonatebackbone, a phosphorothioate backbone, a methylphosphonate backbone, analkylphosphonate backbone, a phosphate ester backbone, analkylphosphonothioate backbone, a phosphorodithioate backbone, acarbonate backbone, a phosphate triester backbone, a carboxymethyl esterbackbone, a methylphosphorothioate backbone, a phosphorodithioatebackbone, a backbone having p-ethoxy linkages, sugar modifications suchas 2′-O-methyl (2′-O-methylnucleotides), 2′-O-methyloxyethoxy(2′-O-MOE), a 2′-O-alkyl modified sugar moiety, or a bicyclic sugarmoiety, nucleotide mimetics, peptide nucleic acids (PNA), morpholinonucleic acids, cyclohexenyl nucleic acids, anhydrohexitol nucleic acids,glycol nucleic acid, threose nucleic acid, and locked nucleic acids(LNA), and any combinations of two or more of any of the foregoing. Inanother aspect, at least one or more nucleotide(s) in the SM-ASOcontains a non-naturally occurring modification to the nucleotide bases.In another aspect, the SM-ASO enhances the degradation of IL7R mRNAsthat lack exon 6 by targeting an IL7R exon 5-exon 7 boundary, e.g., withASOs, siRNAs, shRNAs that decrease stability of sIL7R RNA (e.g.,increase degradation), and/or ASOs that decrease translation of sIL7RRNA. In another aspect, the SM-ASO blocks the translation of IL7R mRNAsthat lack exon 6. In another aspect, the SM-ASO is selected from any ofthe SEQ IDs in Table 1 (SEQ ID NOS:1-13), or portions thereof eitheralone or in combination, or a sequence having at least 70, 75, 80, 84,85, 88, 92, 93, 94, 95, or 96% identity over the full target sequencewithin IL7R RNAs. In another aspect, the oligonucleotide targets any ofthe SEQ IDs in Table 1 (SEQ ID NOS:1-13), either fully or partially. Inanother aspect, the composition further comprises a pharmaceuticallyacceptable excipient, salts, or carrier. In another aspect, theautoimmune disorder is selected from at least one of the following:multiple sclerosis, type I diabetes, rheumatoid arthritis, systemiclupus erythematosus, atopic dermatitis, ankylosing spondylitis, primarybiliary cirrhosis, inflammatory bowel syndromes such as ulcerativecolitis and Crohn's disease, or any conditions where sIL7R is elevated.In another aspect, the method further comprises steps of obtaining cellsfrom the patient and modifying the cells to transiently or permanentlyexpress the oligonucleotide that specifically binds to a sequence of theInterleukin-7 receptor (IL7R) pre-mRNA that influences splicing of exon6. In another aspect, the method further comprises generating a vectorthat expresses the oligonucleotide that specifically binds to a sequenceof the Interleukin-7 receptor (IL7R) pre-mRNA that influences splicingof exon 6 for use in gene therapy, and treating the patient with thevector.

In another embodiment, the present invention includes a composition forincreasing inclusion of exon 6 in an Interleukin-7 receptor (IL7R)pre-mRNA, the method comprising: contacting a splice modulatingantisense oligonucleotide (SM-ASO) that specifically binds to a sequencein the Interleukin-7 receptor (IL7R) pre-mRNA that influences splicingof exon 6, wherein the SM-ASO increases inclusion of exon 6 in IL7Rpre-mRNAs and decreases expression of the soluble isoform of IL7R(sIL7R). In one aspect, the composition further comprises a combinationtherapy of the SM-ASO with one or more active agents effective fortreating autoimmune diseases selected from, but not limited to,mitoxatrone, interferon beta-1a, PEG- interferon beta-1a, azathioprine,fingolimod, natalizumab, methylprednisolone, or ocrelizumab.

In another embodiment, the present invention includes a vector thatexpresses a nucleic acid comprising an oligonucleotide that is anantisense oligonucleotide (ASO), or a splice-modulating antisenseoligonucleotide (SM-ASO), that specifically binds to a sequence in theInterleukin-7 receptor (IL7R) pre-mRNA that influences splicing of exon6, wherein the SM-ASO increases inclusion of exon 6 in IL7R pre-mRNAsand decreases expression of the soluble isoform of IL7R (sIL7R). In oneaspect, the vector is a viral vector or a plasmid.

In another embodiment, the present invention includes a vector thatexpresses a nucleic acid comprising an antisense oligonucleotide (ASO),splice-modulating antisense oligonucleotide (SM-ASO),translation-blocking antisense oligonucleotide, siRNA, shRNA or miRNA,that specifically binds a sequence in the Interleukin-7 receptor (IL7R)pre-mRNA that enhances inhibition or degradation of IL7R RNAs lackingexon 6, wherein the nucleic acid decreases expression of the solubleisoform of IL7R (sIL7R). In one aspect, the vector is a viral vector ora plasmid.

In another embodiment, the present invention includes a method oftreating multiple sclerosis in a subject in need thereof, the methodcomprising: administering an effective amount of a compositioncomprising a splice modulating antisense oligonucleotide (SM-ASO) thatspecifically binds a sequence of an Interleukin-7 receptor (IL7R)pre-mRNA that influences splicing of exon 6, wherein the SM-ASOincreases inclusion of exon 6 in IL7R pre-mRNAs and decreases expressionof the soluble isoform of IL7R (sIL7R) in a pharmaceutically acceptableexcipient. In one aspect, the method further comprises a combinationtherapy of the SM-ASO and one or more active agents effective fortreating multiple sclerosis disease. In another aspect, the one or moreagents for treating multiple sclerosis are selected from, but notlimited to, mitoxatrone, interferon beta-1a, PEG- interferon beta-1a,azathioprine, fingolimod, natalizumab, methylprednisolone, orocrelizumab.

In another embodiment, the present invention includes a method oftreating a cancer in a subject in need thereof, the method comprising:administering an effective amount of a composition comprising anoligonucleotide that specifically binds a sequence of the Interleukin-7receptor (IL7R) pre-mRNA that influences splicing of exon 6, wherein theoligonucleotide decreases inclusion of exon 6 in IL7R pre-mRNAs andincreases expression of the soluble isoform of IL7R (sIL7R). In oneaspect, the oligonucleotide is an antisense oligonucleotide (ASO) or asplice-modulating antisense oligonucleotide (SM-ASO). In another aspect,the oligonucleotide in the composition specifically binds to a sequencein IL7R pre-mRNA in at least one of the group consisting of an exonicsplicing enhancer (ESE) and/or an intronic splicing enhancer (ISE),thereby decreasing inclusion of exon 6, and increasing expression ofsIL7R. In another aspect, the oligonucleotide in the compositionspecifically binds to a sequence on IL7R pre-mRNA at intron-exon splicesites, branchpoint sequences, and/or polypyrimidine tracts. In anotheraspect, at least one or more nucleotide(s) in the oligonucleotidecontains a non-naturally occurring modification comprising modificationsor substitutions of: (1) the ribose or other sugar units, (2) bases, or(3) the backbone, selected from: one or more phosphorothioate,phosphorodithioate, phosphodiester, methyl phosphonate, phosphoramidate,methylphosphonate, phosphotriester, phosphoroaridate, morpholino,amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate,sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilylsubstitutions, partially or completely modified backbones, such as fullymodified sugar phosphate backbone, a locked nucleic acid backbone, apeptidic backbone, a phosphotriester backbone, a phosphoramidatebackbone, a siloxane backbone, a carboxymethylester backbone, anacetamidate backbone, a carbamate backbone, a thioether backbone, abridged methylene phosphonate backbone, a phosphorothioate backbone, amethylphosphonate backbone, an alkylphosphonate backbone, a phosphateester backbone, an alkylphosphonothioate backbone, a phosphorodithioatebackbone, a carbonate backbone, a phosphate triester backbone, acarboxymethyl ester backbone, a methylphosphorothioate backbone, aphosphorodithioate backbone, a backbone having p-ethoxy linkages, sugarmodifications such as 2′-O-methyl (2′-O-methylnucleotides),2′-O-methyloxyethoxy (2′-O-MOE), a 2′-O-alkyl modified sugar moiety, ora bicyclic sugar moiety, nucleotide mimetics, peptide nucleic acids(PNA), morpholino nucleic acids, cyclohexenyl nucleic acids,anhydrohexitol nucleic acids, glycol nucleic acid, threose nucleic acid,and locked nucleic acids (LNA), and combinations of two or more of anyof the foregoing. In another aspect, at least one or more nucleotide(s)in the oligonucleotide contains a non-naturally occurring modificationto the nucleotide bases. In another aspect, the oligonucleotide isselected from any of the SEQ IDs in Table 2 (SEQ ID NOS:14-50), orportions thereof, either alone or in combination, or a sequence havingat least 70, 75, 80, 84, 85, 88, 92, 93, 94, 95, or 96% identity overthe full target sequences within IL7R RNAs. In another aspect, theoligonucleotide targets any of the SEQ IDs in Table 2 (SEQ IDNOS:14-50), or portions thereof. In another aspect, the compositionfurther comprises a pharmaceutically acceptable excipient, salts, orcarrier. In another aspect, the cancer demonstrates low response toconventional immunotherapy (e.g., hepatocellular carcinoma). In anotheraspect, the method further comprises a combination therapy of the SM-ASOand one or more active agents effective for treating cancer such as, butnot limited to, immune check point inhibitors (e.g., nivolumab),therapeutic antibodies (e.g., Herceptin), conventional chemotherapy(e.g., taxol), or therapeutic radiation. In another aspect, the methodfurther comprises steps of obtaining cells from the patient andmodifying the cells to transiently or permanently express theoligonucleotide that specifically binds to a sequence of theInterleukin-7 receptor (IL7R) pre-mRNA that influences splicing of exon6. In another aspect, the method further comprises generating a vectorthat expresses the oligonucleotide that specifically binds to a sequenceof the Interleukin-7 receptor (IL7R) pre-mRNA that influences splicingof exon 6 for use in gene therapy, and treating the patient with thevector.

In another embodiment, the present invention includes a compositioncomprising an oligonucleotide that is an antisense oligonucleotide (ASO)or a splice-modulating antisense oligonucleotide (SM-ASO), thatspecifically binds to a sequence in interleukin 7 receptor (IL7R)pre-mRNA that influences splicing of exon 6, wherein the SM-ASOdecreases inclusion of exon 6 in IL7R pre-mRNAs and increases expressionof the soluble isoform of IL7R (sIL7R). In another aspect, theoligonucleotide in the composition specifically binds to a sequence inIL7R pre-mRNA in at least one of the group consisting of an exonicsplicing enhancer (ESE) and/or an intronic splicing enhancer (ISE),thereby decreasing inclusion of exon 6, and increasing expression ofsIL7R. In another aspect, the oligonucleotide in the compositionspecifically binds to a sequence on IL7R pre-mRNA at intron-exon splicesites, branchpoint sequences, and/or polypyrimidine tracts. In anotheraspect, at least one or more nucleotide(s) in the oligonucleotidecontains a non-naturally occurring modification comprising modificationsor substitutions of: (1) the ribose or other sugar units, (2) bases, or(3) the backbone, selected from: one or more phosphorothioate,phosphorodithioate, phosphodiester, methyl phosphonate, phosphoramidate,methylphosphonate, phosphotriester, phosphoroaridate, morpholino,amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate,sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilylsubstitutions, partially or completely modified backbones, such as fullymodified sugar phosphate backbone, a locked nucleic acid backbone, apeptidic backbone, a phosphotriester backbone, a phosphoramidatebackbone, a siloxane backbone, a carboxymethylester backbone, anacetamidate backbone, a carbamate backbone, a thioether backbone, abridged methylene phosphonate backbone, a phosphorothioate backbone, amethylphosphonate backbone, an alkylphosphonate backbone, a phosphateester backbone, an alkylphosphonothioate backbone, a phosphorodithioatebackbone, a carbonate backbone, a phosphate triester backbone, acarboxymethyl ester backbone, a methylphosphorothioate backbone, aphosphorodithioate backbone, a backbone having p-ethoxy linkages, sugarmodifications such as 2′-O-methyl (2′-O-methylnucleotides),2′-O-methyloxyethoxy (2′-O-MOE), a 2′-O-alkyl modified sugar moiety, ora bicyclic sugar moiety, nucleotide mimetics, peptide nucleic acids(PNA), morpholino nucleic acids, cyclohexenyl nucleic acids,anhydrohexitol nucleic acids, glycol nucleic acid, threose nucleic acid,and locked nucleic acids (LNA), and any combinations of two or more ofany of the foregoing. In another aspect, at least one or morenucleotide(s) in the oligonucleotide contains a non-naturally occurringmodification to the nucleotide bases. In another aspect, theoligonucleotide is selected from any of the SEQ IDs in Table 2 (SEQ IDNOS:14-50), or portions thereof, either alone or in combination, or asequence having at least 70, 75, 80, 84, 85, 88, 92, 93, 94, 95, or 96%identity over the full target sequence within IL7R RNAs. In anotheraspect, the oligonucleotide targets any of the SEQ IDs in Table 2 (SEQID NOS:14-50), either fully or partially. In another aspect, thecomposition further comprises a pharmaceutically acceptable excipient,salts, or carrier. In another aspect, the composition is adapted foradministration to treat a cancer.

In yet another embodiment, the present invention includes a method ofdecreasing inclusion of exon 6 in Interleukin-7 receptor (IL7R)pre-mRNA, the method comprising: contacting a splice modulatingantisense oligonucleotide (SM-ASO) that specifically binds to a sequenceof the Interleukin-7 receptor (IL7R) pre-mRNA that influences splicingof exon 6, wherein the SM-ASO decreases inclusion of exon 6 in IL7Rpre-mRNAs and increases expression of the soluble isoform of IL7R(sIL7R). In another aspect, the oligonucleotide in the compositionspecifically binds to a sequence in IL7R pre-mRNA in at least one of thegroup consisting of an exonic splicing enhancer (ESE) and/or an intronicsplicing enhancer (ISE), thereby decreasing inclusion of exon 6, andincreasing expression of sIL7R. In another aspect, the SM-ASO in thecomposition specifically binds to a sequence on IL7R pre-mRNA atintron-exon splice sites, branchpoint sequences, and/or polypyrimidinetracts. In another aspect, at least one or more nucleotide(s) in theSM-ASO contains a non-naturally occurring modification comprisingmodifications or substitutions of: (1) the ribose or other sugar units,(2) bases, or (3) the backbone, selected from: one or morephosphorothioate, phosphorodithioate, phosphodiester, methylphosphonate, phosphoramidate, methylphosphonate, phosphotriester,phosphoroaridate, morpholino, amidate carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and/or alkylsilyl substitutions, partially or completelymodified backbones, such as fully modified sugar phosphate backbone, alocked nucleic acid backbone, a peptidic backbone, a phosphotriesterbackbone, a phosphoramidate backbone, a siloxane backbone, acarboxymethylester backbone, an acetamidate backbone, a carbamatebackbone, a thioether backbone, a bridged methylene phosphonatebackbone, a phosphorothioate backbone, a methylphosphonate backbone, analkylphosphonate backbone, a phosphate ester backbone, analkylphosphonothioate backbone, a phosphorodithioate backbone, acarbonate backbone, a phosphate triester backbone, a carboxymethyl esterbackbone, a methylphosphorothioate backbone, a phosphorodithioatebackbone, a backbone having p-ethoxy linkages, sugar modifications suchas 2′-O-methyl (2′-O-methylnucleotides), 2′-O-methyloxyethoxy(2′-O-MOE), a 2′-O-alkyl modified sugar moiety, or a bicyclic sugarmoiety, nucleotide mimetics, peptide nucleic acids (PNA), morpholinonucleic acids, cyclohexenyl nucleic acids, anhydrohexitol nucleic acids,glycol nucleic acid, threose nucleic acid, and locked nucleic acids(LNA), and any combinations of two or more of any of the foregoing. Inanother aspect, at least one or more nucleotide(s) in the SM-ASOcontains a non-naturally occurring modification to the nucleotide bases.In another aspect, the SM-ASO enhances the stability of IL7R mRNAs thatlack exon 6 by targeting an IL7R exon 5-exon 7 boundary. In anotheraspect, the SM-ASO enhances the translation of IL7R mRNAs that lack exon6. In another aspect, the SM-ASO is selected from any of the SEQ IDs inTable 2, or portions thereof, either alone or in combination, or asequence having at least 70, 75, 80, 84, 85, 88, 92, 93, 94, 95, or 96%identity over the full target sequence within IL7R RNAs. In anotheraspect, the oligonucleotide targets any of the SEQ IDs in Table 2,either fully or partially. In another aspect, the composition furthercomprises a pharmaceutically acceptable excipient, salts, or carrier. Inanother aspect, the disorder is a type of cancer. In another aspect, themethod further comprises steps of obtaining cells from the patient andmodifying the cells to transiently or permanently express theoligonucleotide that specifically binds to a sequence of theInterleukin-7 receptor (IL7R) pre-mRNA that influences splicing of exon6. In another aspect, the method further comprises generating a vectorthat expresses the oligonucleotide that specifically binds to a sequenceof the Interleukin-7 receptor (IL7R) pre-mRNA that influences splicingof exon 6 for use in gene therapy, and treating the patient with thevector.

In another embodiment, the present invention includes a composition forincreasing inclusion of exon 6 in an Interleukin-7 receptor (IL7R)pre-mRNA, the method comprising: contacting a splice modulatingantisense oligonucleotide (SM-ASO) that specifically binds to a sequencein the Interleukin-7 receptor (IL7R) pre-mRNA that influences splicingof exon 6, wherein the SM-ASO decreases inclusion of exon 6 in IL7Rpre-mRNAs and increases expression of the soluble isoform of IL7R(sIL7R). In one aspect, the composition further comprises a combinationtherapy of the SM-ASO with one or more active agents effective fortreating cancer, such as, but not limited to, immune check pointinhibitors (e.g., nivolumab), therapeutic antibodies (e.g., Herceptin),conventional chemotherapy (e.g., taxol), or therapeutic radiation.

In another embodiment, the present invention includes a vector thatexpresses a nucleic acid comprising an oligonucleotide that is anantisense oligonucleotide (ASO), or a splice-modulating antisenseoligonucleotide (SM-ASO), that specifically binds a sequence in theInterleukin-7 receptor (IL7R) pre-mRNA that influences splicing of exon6, wherein the SM-ASO decreases inclusion of exon 6 in IL7R pre-mRNAsand increases expression of the soluble isoform of IL7R (sIL7R). In oneaspect, the vector is a viral vector or a plasmid.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIGS. 1A to 1C show validation of a GFP-IL7R fluorescent splicingreporter for screening of splice-modulating antisense oligonucleotides(SM-ASOs).

FIGS. 2A to 2E show the targeted screening of IL7R splice-modulatingantisense oligonucleotides (SM-ASOs) complementary to sequences in exon6.

FIGS. 3A to 3E show an SM-ASO walk screen targeting sequences in IL7Rintrons 5 and 6.

FIGS. 4A to 4D show the effects of selected SM-ASOs on expression ofIL7R protein isoforms.

FIGS. 5A to 5D show the dose-response effects of lead SM-ASOs thatreduce sIL7R on modulation of IL7R exon 6 splicing.

FIGS. 6A to 6D show the dose-response effects of lead SM-ASOs thatincrease sIL7R on modulation of IL7R exon 6 splicing.

FIGS. 7A to 7B show correction by IL7R-005 of the effects of the geneticanomaly that increases exclusion of IL7R exon 6 and sIL7R levels in MS.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not limit the invention, except as outlined in the claims.

The present invention is directed to novel compositions and methods thatreduce or increase soluble IL7R (sIL7R) to treat autoimmune diseases(e.g., multiple sclerosis) or cancer, respectively. The presentinvention uses SM-ASOs to control alternative splicing of theInterleukin 7 receptor (IL7R) pre-mRNAs, either to prevent or diminishexpression of sIL7R or the opposite to increase expression of sIL7R. Forexample, given the ability of sIL7R to enhance self-reactivity it isdemonstrated herein that increasing sIL7R levels enhances response tocurrently employed immunotherapies (e.g., immune check pointinhibitors).

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry, organic synthesis,nucleic acid chemistry and nucleic acid hybridization are those wellknown and commonly employed in the art. Further, standard techniques canbe used for nucleic acid and peptide synthesis. Such techniques andprocedures are generally performed according to conventional methodsknown in the art and from various general references (e.g., Sambrook andRussell, 2012, Molecular Cloning, A Laboratory Approach, Cold SpringHarbor Press, Cold Spring Harbor, N.Y., and Ausubel et al., 2012,Current Protocols in Molecular Biology, John Wiley & Sons, NY), relevantportions incorporated herein by reference.

Conventional notations are used herein to describe polynucleotidesequences, e.g., the left-hand end of a single-stranded polynucleotidesequence is the 5′-end and vice versa for the 3′-end (right-hand end);the left-hand direction of a double-stranded polynucleotide sequence isreferred to as the 5′-direction and vice versa for the 3′-direction(right-hand direction), with regard to sequences, such as those thatbecome coding sequences. The direction of 5′ to 3′ addition ofnucleotides to nascent RNA transcripts is referred to as thetranscription direction. The DNA strand having the same sequence as anmRNA is referred to as the “coding strand”. Sequences on the DNA or RNAstrand that are located 5′ to a reference point on the DNA or RNA arereferred to as “upstream sequences”, and sequences on the DNA or RNAstrand that are 3′ to a reference point on the DNA or RNA are referredto as “downstream sequences.”

As used herein, the term “antisense” refers to an oligonucleotide havinga sequence that hybridizes to a target sequence in RNA by Watson-Crickbase pairing, to form an RNA:oligonucleotide heteroduplex with thetarget sequence, typically with an mRNA or pre-mRNA. The antisenseoligonucleotide may have exact sequence complementarity to the targetsequence or near complementarity. These antisense oligonucleotides mayblock or inhibit translation of the mRNA, modify the processing of anmRNA to produce a splice variant of the mRNA, and/or promote specificdegradation of a given mRNA or variant of an mRNA. One non-limitingexample can also be RNase H dependent degradation. It is not necessarythat the antisense sequence be complementary solely to the codingportion of the RNA molecule. The antisense sequence may be complementaryto regulatory sequences specified on the non-coding region of an RNAmolecule (e.g. introns, untranslated regions) encoding a protein, whichregulatory sequences control expression of the coding sequences.Antisense oligonucleotides are typically between about 5 to about 100nucleotides in length, more typically, between about 7 and about 50nucleotides in length, and even more typically between about 10nucleotides and about 30 nucleotides in length.

As used herein, the term “nucleic acid” or a “nucleic acid molecule”refer to any DNA or RNA molecule, either single or double stranded,whether in linear or circular form. With reference to nucleic acids ofthe present invention, the term “isolated nucleic acid”, when applied toDNA or RNA, refers to a DNA or RNA molecule that is separated fromsequences with which it is immediately contiguous in the naturallyoccurring genome or gene products of the organism in which itoriginated. For example, an “isolated nucleic acid” may comprise a DNAmolecule inserted into a vector, such as a plasmid or virus vector, orintegrated into the genomic DNA of a prokaryotic or eukaryotic cell orhost organism.

As used herein, the terms “specifically hybridizing” or “substantiallycomplementary” refer to the association between two nucleotide moleculesof sufficient complementarity to permit hybridization underpre-determined conditions generally used in the art. Examples of low,middle or intermediate and high stringency hybridization conditions arewell known to the skilled artisan, e.g., using Sambrook and Russell,2012, Molecular Cloning, A Laboratory Approach, Cold Spring HarborPress, Cold Spring Harbor, N.Y., or Ausubel et al., 2012, CurrentProtocols in Molecular Biology, John Wiley & Sons, NY, relevant portionsincorporated herein by reference.

As used herein, the phrase “chemically modified oligonucleotide” refersto a short nucleic acid (DNA or RNA) that can be a sense or antisensethat includes modifications or substitutions, such as those taught byWan and Seth, “The Medicinal Chemistry of Therapeutic Oligonucleotides”,J. Med. Chem. 2016, 59, 21, 9645-9667, relevant portions incorporatedwherein, which may include modifications of: (1) the ribose or othersugar units, (2) bases, or (3) the backbone, which in nature is composedof phosphates, as are known in the art. Non-limiting examples ofmodifications or nucleotide analogs include, without limitation,nucleotides with phosphate modifications comprising one or morephosphorothioate, phosphorodithioate, phosphodiester, methylphosphonate, phosphoramidate, methylphosphonate, phosphotriester,phosphoroaridate, morpholino, amidate carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and/or alkylsilyl substitutions (see, e.g., Hunziker andLeumann (1995) Nucleic Acid Analogues: Synthesis and Properties, inModern Synthetic Methods, VCH, 331-417; Mesmaeker et al. (1994) NovelBackbone Replacements for Oligonucleotides, in CarbohydrateModifications in Antisense Research, ACS, 24-39); nucleotides withmodified sugars (see, e.g., U.S. Patent Application Publication No.2005/0118605) and sugar modifications such as 2′-O-methyl(2′-O-methylnucleotides) and 2′-O-methyloxyethoxy (2′-O-MOE), a2′-O-alkyl modified sugar moiety, or a bicyclic sugar moiety, andnucleotide mimetics such as, without limitation, peptide nucleic acids(PNA), morpholino nucleic acids, cyclohexenyl nucleic acids,anhydrohexitol nucleic acids, glycol nucleic acid, threose nucleic acid,and locked nucleic acids (LNA), as well as partially or completelymodified backbones, such as fully modified sugar phosphate backbone, alocked nucleic acid backbone, a peptidic backbone, a phosphotriesterbackbone, a phosphoramidate backbone, a siloxane backbone, acarboxymethylester backbone, an acetamidate backbone, a carbamatebackbone, a thioether backbone, a bridged methylene phosphonatebackbone, a phosphorothioate backbone, a methylphosphonate backbone, analkylphosphonate backbone, a phosphate ester backbone, analkylphosphonothioate backbone, a phosphorodithioate backbone, acarbonate backbone, a phosphate triester backbone, a carboxymethyl esterbackbone, a methylphosphorothioate backbone, a phosphorodithioatebackbone, a backbone having p-ethoxy linkages, and a combinations of twoor more of any of the foregoing (see, e.g., U.S. Pat. Nos. 5,886,165;6,140,482; 5,693,773; 5,856,462; 5,973,136; 5,929,226; 6,194,598;6,172,209; 6,175,004; 6,166,197; 6,166,188; 6,160,152; 6,160,109;6,153,737; 6,147,200; 6,146,829; 6,127,533; and 6,124,445, relevantportions incorporated herein by reference).

As used herein, the term “expression cassette” refers to a nucleic acidmolecule comprising a coding sequence operably linked topromoter/regulatory sequences necessary for transcription, processingand, optionally, translation or splicing of the coding sequence.

The IL7R SM-ASOs that decrease sIL7R can be used for the treatment ofdiseases or disorders such as autoimmune and/or inflammatory diseases.The IL7R SM-ASOs that increase sIL7R can be used for immuno-oncologyapplications. Whether increasing or decreasing the expression of sIL7Rmessage or protein, the present invention can used be used inconjunction with gene therapy and ex vivo applications. For example, theoligonucleotides can be used in a method in which cells are isolatedfrom the subject or another subject, and the cells are modified toexpress the oligonucleotides that modify the expression of sIL7R, andthe cells can then be transferred back into the subject. The presentinvention can be used with the various known methods of delivery andexpression, such as plasmid or viral vectors. Also, the presentinvention can be used with all methods for modification of cells, e.g.,gene editing, delivery of nucleic acids (any nucleic acid, eithernatural, synthetic or modified), proteins (full-length protein orpeptides), whether transient or permanent, or under the control ofregulatable promoters. The oligonucleotide or vectors that express thesame can be delivered via known methods, such as, e.g., transfection,electroporation, carrier-mediated, viral, etc.

As used herein, the term “promoter/regulatory sequence” refers to anucleic acid sequence that is required for expression of a gene productoperably linked to the promoter/regulator sequence. In some instances,the promoter/regulatory sequence may be the core promoter sequence andin other instances, this sequence may also include an enhancer sequenceand other regulatory elements that are required for expression of thegene product. The promoter/regulatory sequence may be, for example, asequence that drives the expression of a gene product in a constitutiveand/or inducible manner. As used herein, the term “inducible promoter”refers to a nucleotide sequence which, when operably linked with apolynucleotide which encodes or specifies a gene product, causes thegene product to be produced substantially only when an inducer whichcorresponds to the promoter is present.

As used herein, the terms “percent similarity”, “percent identity” and“percent homology”, when referring to a comparison between two specificsequences, identify the percentage or bases that are the same along aparticular sequence. The percentage of similarity, identity or homologycan be calculated using, e.g., the University of Wisconsin GCG softwareprogram or equivalents.

As used herein, the term “replicon” refers to any genetic element, forexample, a plasmid, cosmid, bacmid, phage or virus, which is capable ofreplication largely under its own control. A replicon may be either RNAor DNA and may be single or double stranded.

As used herein, the term “vector” refers to a genetic element, such as aplasmid, cosmid, bacmid, phage or virus, to which another geneticsequence or element (either DNA or RNA) may be attached. The vector maybe a replicon so as to bring about the replication of the attachedsequence or element. An “expression vector” is a vector that facilitatesthe expression of a nucleic acid, such as an oligonucleotide, or apolypeptide coding nucleic acid sequence in a host cell or organism.

As used herein, the term “operably linked” refers to a nucleic acidsequence placed into a functional relationship with another nucleic acidsequence. Examples of nucleic acid sequences that may be operably linkedinclude, without limitation, promoters, transcription terminators,enhancers or activators and heterologous genes which when transcribedand, if appropriate to, translated will produce a functional productsuch as a protein, ribozyme or RNA molecule.

As used herein, the term “oligonucleotide,” refers to a nucleic acidstrand, single or double stranded that has a length that is, typically,less than a coding sequence for a gene, e.g., the oligonucleotide willgenerally be at least 4-6 bases or base-pairs in length, and up to about200, with the most typical oligonucleotide being in the range of 8-20,10-25, 12-30, or about 30, 35, 40, or 50 bases or base-pairs. In onespecific example of the present invention, the oligonucleotide is anucleic acid strand having a sequence that modulates the inclusion ofexon 6 in pre-mRNAs of the Interleukin-7 receptor (IL7R) gene, and isdefined as a nucleic acid molecule comprised of two or more ribo ordeoxyribonucleotides, preferably more than four. The exact size of theoligonucleotide will depend on various factors and on the particularapplication and use of the oligonucleotide, which can be varied as willbe known to the skilled artisan without undue experimentation followingthe teachings herein and as taught in, e.g., Sambrook and Russell, 2012,Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, ColdSpring Harbor, N.Y., or Ausubel et al., 2012, Current Protocols inMolecular Biology, John Wiley & Sons, NY, relevant portions incorporatedherein by reference.

As used herein, the term “splice variant or isoform of an mRNA”, ismeant a variant mRNA, which could be defective or pathogenic and be theresult of alternative splicing of the RNA encoding a protein. Splicingevents that produce a splice variant of the mRNA that is defective orleads to pathology will be referred in the present invention as asplicing defect. One example of such a splicing defect is the exclusionof exon 6 of IL7R causing expression of a shorter protein, soluble IL7R(sIL7R), which is secreted from the cell, leading to its presence in,e.g., the bloodstream or other bodily fluids. The present inventiontargets the elements (i.e., sequences) within IL7R pre-mRNAs thatcontrol the inclusion or exclusion of IL7R exon 6 in the final mature orprocessed mRNA, or sequences within sIL7R mRNA that control itstranslation or stability.

As used herein, the term “splice variant or isoform of a protein”, ismeant a variant protein, which could be defective or pathogenic and bethe result of alternative splicing of an RNA encoding a protein.Alternatively, when discussing those variants that increase degradation,those splice variants would reduce or eliminate protein production.Splicing events that produce a splice variant of a protein that isdefective or leads to pathology will be referred in the presentinvention as a splicing defect. One example of such a splicing defect isthe exclusion of exon 6 of IL7R causing expression of a shorter protein,soluble IL7R (sIL7R), which is secreted from the cell, leading to itspresence in, e.g., the bloodstream or other bodily fluids. The presentinvention targets the elements (i.e., sequences) within IL7R pre-mRNAsthat control the inclusion or exclusion of IL7R exon 6 in the finalmature or processed mRNA, or sequences within sIL7R mRNA that controlits translation or stability.

As used herein, the term “treatment”, refers to reversing, alleviating,delaying the onset of, inhibiting the progress of, and/or preventing adisease or disorder, or one or more symptoms thereof, to which the termis applied in a subject, e.g., an autoimmune disease or disorder. Insome embodiments, the treatment may be applied after one or moresymptoms have developed. In other embodiments, treatment may beadministered in the absence of symptoms. For example, treatment may beadministered prior to symptoms (e.g., in light of a history of symptomsand/or one or more other susceptibility factors), or after symptoms haveresolved, for example to prevent or delay their reoccurrence. One suchnon-limiting example is relapsing-remitting MS.

As used herein, the terms “effective amount” and “pharmaceuticallyeffective amount” refer to a sufficient amount of an agent to providethe desired biological result. Preferably, the sufficient amount of theagent does not induce toxic side effects. The present invention usingIL7R SM-ASOs that reduce sIL7R should lead to a reduction and/oralleviation of the signs, symptoms, or causes of autoimmune diseases ordisorders. As designed, the present invention is not expected to cause areduction in the host immune response, and thus have few or low sideeffects associated with broad immunosuppression. An appropriateeffective amount in any individual case may be determined by one ofordinary skill in the art using routine experimentation. The presentinvention using IL7R SM-ASOs that increase sIL7R should lead to areduction and/or alleviation of the signs, symptoms, or causes ofcancers. As designed, the present invention is expected to cause anenhancement in the host immune response and thus to enhance currentimmunotherapies. An appropriate effective amount in any individual casemay be determined by one of ordinary skill in the art using routineexperimentation.

The present invention may be provided in conjunction with one or more“pharmaceutically acceptable” agents, carriers, buffers, salts, or otheragents listed in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans, whichgenerally indicates approval by a regulatory agency of the Federalgovernment or a state government. Typical pharmaceutically acceptableformulations for use with oligonucleotides include but are not limitedto salts such as: calcium chloride dihydrate (US Pharmacopeia (USP)),magnesium chloride hexahydrate USP, potassium chloride USP, sodiumchloride USP; and may include buffers such as” sodium phosphate dibasicanhydrous USP, sodium phosphate monobasic dihydrate USP, and water USP.Typically, the pH of the product may be modified using hydrochloric acidor sodium hydroxide to a pH of ˜6.8, 6.9, 7.0, 7.1, or 7.2.

The present invention may be provided in conjunction with one or morediagnostic tests that are used to demonstrate the effectiveness of thetreatment.

As used herein, the term “carrier” refers to, for example, a diluent,preservative, solubilizer, emulsifier, adjuvant, excipient, auxiliaryagent or vehicle with which an active agent of the present invention isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water or aqueous saline solutions and aqueous dextroseand glycerol solutions may be employed as carriers. Suitablepharmaceutical carriers are described in “Remington's PharmaceuticalSciences” 2012.

In one embodiment, the present invention is directed to novelcompositions and methods to treat autoimmune disorders, including butnot limited to Multiple Sclerosis (MS). MS is the most commonneurological disease of early adulthood and is mediated by autoimmunemechanisms that lead to demyelination and neuronal damage in the centralnervous system, resulting in progressive neurological dysfunction. Todate, there is no cure for the disease and current available treatmentsfocus on preventing future immunological attacks, often by suppressingthe immune system. This immunosuppressive approach causes a plethora ofadverse side effects, among them increased risk of cancer and infectionsthat could be severe or lethal. Accordingly, the present inventors havedeveloped novel therapeutics to meet the clear, unmet need for thedevelopment of effective and well-tolerated therapies to arrest MSdevelopment, without adverse immunosuppressive side effects.

The present invention is also directed to novel compositions and methodsto treat cancer. Cancer is the second cause of death in the UnitedStates and is mediated by many etiologies. To date, many cancers cannotbe treated; however, recently therapies based on immune recognition andkilling of cancer cells have led to new hope. Unfortunately, manypatients receiving immunotherapy do not respond well and some cancertypes (e.g., hepatocellular carcinoma) have low response rates.Accordingly, the present inventors have developed novel therapeutics tomeet the clear, unmet need for the enhancement of conventionalimmunotherapies.

The present inventors have developed targeted antisenseoligonucleotides, such as antisense oligonucleotides (ASOs) and/orsplice-modulating antisense oligonucleotides (SM-ASOs), to correct aspecific MS etiology. SM-ASOs have proven to be a novel and valuabletherapeutic tools to treat disorders caused by aberrant RNA splicing.Two such SM-ASOs have received recent approvals by the FDA for treatmentof Spinal Muscular Atrophy (Spinraza, Biogen) and Duchenne MuscularDystrophy (Exondys 51, Sarepta Therapeutics).

The novel targeted therapeutics correct aberrant splicing of theinterleukin 7 receptor (IL7R) RNAs, where exclusion of the alternativeexon 6 leads to elevated levels of the pathogenic soluble form of theIL7R (sIL7R). Several lines of evidence directly link and stronglysupport a role for alternative splicing of IL7R exon 6 in thepathogenesis of MS and other autoimmune diseases: (1) genetic variantsthat increase exclusion of this exon are strongly associated withincreased MS risk (Galarza-Munoz et al., 2017; Gregory et al., 2007);(2) sIL7R exacerbates the severity and progression of the disease in theExperimental Autoimmune Encephalomyelitis (EAE) mouse model of MS(Lundstrom et al., 2013); and (3) sIL7R is elevated in patients fromseveral autoimmune diseases including MS, type I diabetes, rheumatoidarthritis and systemic lupus erythematosus (Badot et al., 2011; Badot etal., 2013; Lauwerys et al., 2014; McKay et al., 2008; Monti et al.,2013).

The present inventors have developed several SM-ASOs (Table 1), amongthem the lead ASOs IL7R-005 and IL7R-006, that promote inclusion of exon6 in IL7R pre-mRNAs and correct expression of IL7R protein isoforms incultured cells. Critical to this therapeutic approach, these SM-ASOsdecrease sIL7R levels without a negative impact on expression of themembrane-bound IL7R (mIL7R). To treat autoimmunity, the presentinvention is used to block specific sequences in IL7R pre-mRNAs thatdrive exon 6 exclusion, thereby promoting exon 6 inclusion and reducingsIL7R levels. Additionally, to treat cancer, the present invention isused to block specific sequences in IL7R pre-mRNAs that drive exon 6inclusion, thereby decreasing exon 6 inclusion and increasing sIL7Rlevels. The skilled artisan will recognize that the SM-ASOs of thepresent invention can be used alone or in combination with othertherapies. Furthermore, through simple single base mutation, the SM-ASOscan be adapted to control splicing of exon 6 in allelic variants ofhuman IL7R, or of IL7R in different animals, or in individuals carryingpolymorphisms or where mutations have occurred at the targetedsequences, thus tailoring the complementarity of the SM-ASOs to thevariant sequences.

TABLE 1 Target sequence of SM-ASOs that reduce sIL7R. SEQ Target DiseaseID SM-ASO Sequence* Efficiency{circumflex over ( )} application NO:IL7R-005 GUCGCUCUGUUGGUC +++ Autoimmunity  1 IL7R-006 UAAUAAAGAGGGUGA+++ Autoimmunity  2 UUGUG IL7R-008 UUGUGUGGGAUCACG +++ Autoimmunity  3(Cluster 2) IL7R-009 UGGGAUCACGGACAG +++ Autoimmunity  4 (Cluster 2)IL7R-010 UCACGGACAGUCAGA +++ Autoimmunity  5 (Cluster 2) Cluster 2UUGUGUGGGAUCACG +++ Autoimmunity  6 GACAGUCAGA IL7R-011GACAGUCAGAGCUUA + Autoimmunity  7 AGC IL7R-017 UGAGAAAACCACAAA ++Autoimmunity  8 IL7R-024 UACC C CCACUGCAUG + Autoimmunity  9 IL7R-025GACCCUACC C CCACU +++ Autoimmunity 10 (Cluster 1) IL7R-026CCUGAGACCCUACCC +++ Autoimmunity 11 (Cluster 1) IL7R-027 AGCACCCUGAGACCC+++ Autoimmunity 12 (Cluster 1) Cluster 1 AGCACCCUGAGACCC +++Autoimmunity 13 UACC C CCACU *Nucleotides highlighted in bold andunderlined indicate positions were a mismatch was engineered at thecomplementary position in the SM-ASO to disrupt potential secondarystructures that could limit the activity of the SM-ASO. {circumflex over( )}Efficacy scale: low (+), intermediate (++), and high (+++).

A wide variety of SM-ASOs can be used with the present invention, e.g.,those that include a wide variety of base or backbone modifications tothe SM-ASOs. Non-limiting examples of SM-ASOs can include native nucleicacids, but can also include, e.g., backbone or base modifications(chemically modified oligonucleotides) that increase the efficiency ofbinding of the SM-ASO, increase the stability (e.g., half-life) of theSM-ASO, increase its expression, control where its expressed,distributed or localized, and the like.

Current treatments for autoimmune diseases, such as MS, have helpedautoimmune disease patients manage their symptoms, yet these drugs arefar from ideal given the wide variety of adverse side effects theycause, which can be severe or lethal. The development of effective butyet safer MS drugs has been hindered by the complex nature of thedisease, wherein a multitude of etiologies lead to MS pathogenesis.Given that all these etiologies culminate in a breach of immunologicaltolerance against myelin, the field has, so far, focused on developingtherapies to diminish immunological responses via diverse mechanisms.For example, natalizumab is designed to block migration of leukocytesacross blood-brain barrier and their recruitment to sites ofinflammation. Another example is ocrelizumab, which depletes B-cells.However, both of these mechanisms (although through different actions)ultimately lead to immunosuppression. In order to provide effective yetsafer drugs to the patients, instead of dealing with the consequences ofa given etiology via immune modulation, it is imperative to develop newtherapies targeting correction of specific MS etiologies, which thepresent inventors refer to herein as immune correction.

The canonical, membrane-bound interleukin 7 receptor (mIL7R) has been aprevious candidate of therapeutic intervention in MS and numerousautoimmune disorders. However, mIL7R is crucial for T cell homeostasisand normal immune function, and loss of IL7R function in both human andmouse cause severe immunodeficiency (Maraskovsky et al., 1996; Peschonet al., 1994; Puel et al., 1998; Roifman et al., 2000). Accordingly,novel MS therapies that inhibit expression or function of mIL7R wouldcause severe immunodeficiency. The therapeutic SM-ASOs developed herecorrect aberrant splicing of IL7R exon 6, and in doing so, they diminishsIL7R levels while preventing a negative impact on mIL7R expressionand/or function. Accordingly, unlike current MS treatments relying onimmunosuppressive mechanisms, the therapeutic SM-ASOs of the presentinvention that reduce sIL7R (Table 1) are an effective and safer optionto treat MS that avoid immunosuppression. The SM-ASOs of the presentinvention that reduce sIL7R (Table 1) represent a major improvement overcurrent drugs in that they correct the root of the problem rather thandeal with the consequences of it by suppressing the immune system.

Furthermore, increased levels of sIL7R (when compared to the normallevels of sIL7R in subject that does not have an autoimmune disease) hasbeen associated with other autoimmune diseases such as type I diabetes,rheumatoid arthritis and systemic lupus erythematosus, and patients ofthese diseases have been shown to have elevated levels of circulatingsIL7R (Badot et al., 2011; Badot et al., 2013; Lauwerys et al., 2014;Monti et al., 2013). Therefore, the therapeutic SM-ASOs could be used totreat numerous diseases or disorders that have elevated levels of sIL7R.

Cancer is the second leading cause of death in the United States.Effective treatment options for many cancers are lacking given the manyetiologies that orchestrate it. Immuno-oncology is a rapidly growingfield that uses the body's immune system as novel treatments forpreviously intractable cancers. Immune check point inhibitors, such asmonoclonal antibodies that target the negative immune regulators CTLA-4(Ipilimumab (Yervoy, Bristol-Myers Squibb) and PD-1 (Pembrolizumab(Keytruda, Merck) and Nivolumab (Opdivo, Bristol-Myers Squibb) are FDAapproved. An example their potential is the study in patients withpreviously untreated, and inoperable or metastatic melanoma who weretreated with a combination of Nivolumab and Ipilimumab, which reported a50% overall response rate (Larkin J, Chiarion-Sileni V, Gonzalez R, GrobJ J, Cowey C L, et al. 2015. N Engl J Med 373: 23-34).

Although recent cancer immunotherapies have provided new hope,unfortunately, many patients receiving immunotherapy do not respond welland some cancer types (e.g., hepatocellular carcinoma) have low responserates. For instance, while Nivolumab is FDA-approved for patients withhepatocellular carcinoma who failed to respond to the kinase inhibitorSorafenib, the overall response rate among 154 patients tested was only14.3% and a complete response was observed in only 3 patients(NCT01658878). Although encouraging, these data indicate that there is acritical need to increase the response rate.

The need to increase immunotherapeutic response rate has inspired theintense search for markers (e.g., PD-L1 expression and highmicrosatellite instability in tumor cells) that predict success for thecurrently deployed immune check point blockers. Equally exciting,although less developed, is the search for pro-immune modulators thatcould synergize with check point blockade. Elevated levels of sIL7R arethought to enhance immunological responses by potentiating thebioavailability and/or bioactivity of the cytokine IL7, leading toenhanced survival of T cells (Lundstrom et al., 2013). This creates apro-inflammatory environment that has the potential to increase immuneresponses against cancers. Accordingly, the present invention usesSM-ASOs that increase sIL7R, such as lead SM-ASOs IL7R-001 and IL7R-004,and additional SM-ASOs in Table 2, as a novel immunotherapy againstcancer.

TABLE 2 Target sequence of SM-ASOs that increase sIL7R. SEQ TargetDisease ID SM-ASO Sequence* Efficiency{circumflex over ( )} applicationNO IL7R-001 AGGUGACCUUCUUCA +++ Immuno-oncology 14 ACUAAUAAAG IL7R-002CAUCAGCAUUUUGAG +++ Immuno-oncology 15 IL7R-003 UUUUUUCUCUGUCGC +++Immuno-oncology 16 UCUGUUGGUC IL7R-004 UCUCUGUCGCUCUGU +++Immuno-oncology 17 IL7R-007 GGUGAUUGUGUGGGA + Immuno-oncology 18IL7R-013 GCUUAAGCCCCAUUU + Immuno-oncology 19 AUUGAUG IL7R-015CAUUUAUUGAUGAGA +++ Immuno-oncology 20 AAACCA IL7R-018 AAACCACAAAGGGGA++ Immuno-oncology 21 IL7R-019 ACAAAGGGGAUUAAG +++ Immuno-oncology 22 GIL7R-020 GGGGAUUAAGGCAUU ++ Immuno-oncology 23 UCA IL7R-021UUAAGGCAUUUCACG + Immuno-oncology 24 A IL7R-023 UCACGAAUUUAGUGC +Immuno-oncology 25 C IL7R-031 CCCACAUUACUAAGU + Immuno-oncology 26 AAAIL7R-032 GGCCCACAUUACUAA ++ Immuno-oncology 27 IL7R-033AAGUGGGCCCACAUU + Immuno-oncology 28 IL7R-034 AUAAUAAGUGGGCCC +Immuno-oncology 29 IL7R-039 UCUCUUAACUGAAAA + Immuno-oncology 30 GCAAIL7R-042 CAAAUAUGUCUCUUA +++ Immuno-oncology 31 ACUGAAA IL7R-043UGUCAAAUAUGUCUC ++ Immuno-oncology 32 UUAAC IL7R-044 GCUGUCAAAUAUGUC +Immuno-oncology 33 UC IL7R-045 AUAAAGCUGUCAAAU + Immuno-oncology 34 AUIL7R-046 UCCAUAAAGCUGUCA ++ Immuno-oncology 35 IL7R-047AUCCCUCCAUAAAGC + Immuno-oncology 36 IL7R-048 AACCAAAAUCCCUCC ++Immuno-oncology 37 AU IL7R-049 UGCCUUUUAAACCAA +++ Immuno-oncology 38AAUCCC IL7R-050 GUCAAUGCCUUUUAA ++ Immuno-oncology 39 ACCAAA IL7R-051CCCAAGUCAAUGCCU + Immuno-oncology 40 UUUAAA IL7R-053 GUCACCCAAGUCAAU +Immuno-oncology 41 IL7R-054 GCCUGGUCACCCAAG + Immuno-oncology 42IL7R-058 AAUUUAGUGCCCAGU + Immuno-oncology 43 IL7R-059 AGUGCCCAGUAUCCC++ Immuno-oncology 44 IL7R-060 CCAGUAUCCCUAUCU ++ Immuno-oncology 45IL7R-061 AUCCCUAUCUAUCCU ++ Immuno-oncology 46 CA IL7R-062UAUCUAUCCUCAGCG + Immuno-oncology 47 IL7R-063 AUCCUCAGCGAAUUU +Immuno-oncology 48 C IL7R-064 CAGCGAAUUUCCACA + Immuno-oncology 49IL7R-065 AAUUUCCACAGUUAA ++ Immuno-oncology 50 UUUCAUAAG *Nucleotideshighlighted in bold and underlined indicate positions were a mismatchwas engineered at the complementary position in the SM-ASO to disruptpotential secondary structures that could limit the activity of theSM-ASO. {circumflex over ( )}Efficacy scale: low (+), intermediate (++),and high (+++).

FIGS. 1A to 1C show validation of a GFP-IL7R fluorescent splicingreporter for screening of splice-modulating antisense oligonucleotides(SM-ASOs). FIG. 1A shows schematics of the GFP-IL7R reporterillustrating location of SM-ASO targets (red) and mutations of thecorresponding cis-splicing elements (blue). FIG. 1B shows splicinganalysis of IL7R exon 6 in transcripts from the GFP-IL7R reporter. Helacells stably expressing either WT (C) or mutant versions of the reporter(5′Mut, 5′Cons, ΔESE2 & ΔESS2) were transfected with either control(Ctrl) or experimental (IL7R-001 & IL7R-002) SM-ASOs. Exon 6 splicingwas analyzed by RT-PCR using primers specific for the GFP-IL7R reporter(+E6=exon 6 included; −E6=exon 6 excluded), and percentage of exon 6inclusion was calculated as: [included/(included+excluded)]*100. FIG. 1Cshows analysis of GFP expression. GFP mean fluorescence intensity (MFI)was quantified by flow cytometry. Data are shown normalized to controlSM-ASO (Ctrl).

To assess the feasibility of this reporter system to screen for SM-ASOsthat modulate splicing of IL7R exon 6, the present inventors comparedthe effects of blocking specific cis-acting splicing elements in exon 6with mutation of the corresponding elements. For example, SM-ASOIL7R-001 blocks the 5′-splice site of exon 6 and forces almost completeexclusion of the exon equivalent to a mutation that cripples this5′-splice site (5′Mut). Likewise, blocking of the previously identifiedexonic splicing enhancer 2 (ESE2) by IL7R-002 causes comparable effectsthan mutation of this enhancer (ΔESE2). IL7R-002 has low affinity forIL7R RNAs, which likely explains the slightly lower magnitude comparedto ΔESE2. The fact that SM-ASO-mediated blocking or mutation of a givencis-splicing element caused equivalent effects demonstrates the power ofSM-ASOs to control splicing decisions in this reporter system. Moreimportantly, the observed changes in exon 6 splicing lead to theexpected changes in GFP expression, thereby validating the use of GFPexpression as readout of splicing outcomes in this reporter system.

FIGS. 2A to 2E shows a targeted screening of IL7R SM-ASOs complementaryto cis-acting splicing elements within exon 6. FIG. 2A shows schematicsof the GFP-IL7R splicing fluorescent reporter used for screening. Thegenomic sequence of IL7R spanning introns 5 and 6 was clonedinterrupting the GFP coding sequence, so that GFP expression isdetermined by splicing of IL7R exon 6. FIGS. 2B and 2C show analysis ofGFP expression. HeLa cells stably expressing the fluorescent reporterwere transfected with either control (ASO-Ctrl) or experimentalmorpholino SM-ASOs (IL7R-001-IL7R-005), and GFP expression wasquantified by flow cytometry. FIG. 2B shows representative histograms ofGFP mean fluorescence intensity (MFI) for selected SM-ASOs, whereas FIG.2C shows quantification of GFP MFI normalized to control ASO (ASO-ctrl).Red dashed lines indicate efficacy cutoff of 1.5 fold change in GFPexpression in either direction. FIG. 2D shows splicing analysis of IL7Rexon 6 in transcripts from the GFP-IL7R reporter. Splicing of IL7R exon6 was analyzed in transcripts from the reporter by RT-PCR using primersspecific for the GFP-IL7R reporter (+E6=exon 6 included; −E6=exon 6excluded), and percentage of exon 6 inclusion was determined as:[included/(included+excluded)]*100. Red dashed lines indicate efficacycutoff of 1.5 fold change in percentage of exon 6 inclusion. FIG. 2Eshows splicing analysis of IL7R exon 6 in transcripts from theendogenous IL7R gene. Splicing of IL7R exon 6 was analyzed intranscripts from the endogenous IL7R gene with primers specific for theendogenous transcripts (FL=exon 6 included; ΔE6=exon 6 excluded), andpercentage of exon 6 inclusion was determined as: [FL/(FL+ΔE6)]*100. Inall panels, statistical significance was assessed by two-tailedStudent's t-test comparing experimental ASOs versus control (*p<0.05,**p<0.005, ***p<0.0005).

This targeted screen uncovered several SM-ASOs that modulate splicing ofIL7R exon 6 in either direction. For example, blocking of the 5′-splicesite (IL7R-001) or the previously identified ESE2 (IL7R-002) inducesalmost complete exclusion of the exon. Most importantly, it uncoveredIL7R-005, which blocks the exonic splicing silencer 3 (ESS3) promotingalmost complete exon inclusion. This one SM-ASO was found to beparticularly useful for the treatment of autoimmune diseases. Inaddition, the present inventors uncovered four SM-ASOs that enhance exon6 exclusion (IL7R-001, IL7R-002, IL7R-003 and IL7R-004), which arecandidates for immuno-oncology applications. Critical for therapeuticpurposes, all SM-ASOs tested induced similar modulation of exon 6splicing in transcripts from both the GFP-IL7R reporter and theendogenous IL7R gene.

FIGS. 3A to 3E show an SM-ASO walk screen targeting sequences in IL7Rintrons 5 and 6. FIG. 3A shows the schematics of the SM-ASO walkapproach. As an example, the present inventors designed morpholinoSM-ASOs of 15-25 nt in length complementary to overlapping sequencesevery 5 nt within the intronic regions proximal to IL7R exon 6. Theseregions include the last 209 nt of IL7R intron 5, avoiding the last 40nt which contain core splicing elements such as the branchpointsequence, polypyrimidine tract and 3′-splice site, and the first 169 ntof IL7R intron 6, avoiding the first 15 nt which includes the 5′-splicesite. Control (ASO-Ctrl) or experimental SM-ASOs targeting theseintronic regions (e.g., IL7R-006-IL7R-065) were transfected into HeLacells stably expressing the reporter system as in FIG. 2A. IL7R-001 andIL7R-005 were used as positive controls. FIGS. 3B and 3C show GFPexpression analysis of SM-ASOs targeting introns 5 (FIG. 3B) and 6 (FIG.3C). GFP mean fluorescence intensity (MFI) was determined by flowcytometry as before. Red and blue lines indicate cutoff of 20% decreaseor increase in GFP MFI, respectively. FIGS. 3D and 3E show splicinganalysis of IL7R exon 6 in transcripts from the GFP-IL7R reporter forselected SM-ASOs targeting introns 5 (FIG. 3D) and 6 (FIG. 3E). Thepercentage of exon 6 inclusion in transcripts from the reporter wasdetermined as before. Red dash line indicates the level of exon 6inclusion in the control (ASO-Ctrl). In all panels, statisticalsignificance was assessed by two-tailed Student's t-test comparingexperimental SM-ASOs versus control (*p<0.05, **p<0.005, ***p<0.0005).This approach uncovered IL7R-006, another SM-ASO, which promotes highlevels of exon 6 inclusion by blocking sequences within intron 6. Withinthe target sequence of SM-ASO IL7R-006 (UAAUAAAGAGGGUGAUUGUG) lies apoly adenylation signal (AAUAAA) near the 5′ splice site of intron 6,which the inventors previously found to promote skipping of exon 6, mostlikely by blocking the 5′-splice site when bound by CPSF1 (Evsyukova etal., 2013). However, the IL7R-006 oligonucleotide of the presentinvention blocks a 20 nt sequence that contains additional splicingregulatory sequences, which is different from results published inEvsyukova et al., 2013.

In addition to IL7R-006, the present inventors also uncovered additionaltarget sequences that increase exon 6 inclusion (listed in Table 1).These include two clusters of SM-ASOs, Cluster 1 (intron 5) and Cluster2 (intron 6), and several other SM-ASOs, which increase exon 6 inclusionalbeit less efficiently than lead SM-ASOs IL7R-005 and IL7R-006. Thepresent inventors are currently refining the final target sequence forthe newly discovered SM-ASOs to improve their efficiency.

This screen also uncovered several SM-ASOs that decrease exon 6inclusion (seen as increased GFP expression in FIGS. 3B and 3C, andlisted in Table 2). The latter are candidates for therapeuticintervention in immuno-oncology. Importantly, this approach identifiednew SM-ASO targets for autoimmunity and cancer to be tested inpre-clinical models.

FIGS. 4A to 4D show the effects of selected SM-ASOs on expression ofIL7R protein isoforms. HeLa cells were transfected with control(ASO-Ctrl) or experimental (IL7R-001, IL7R-004, IL7R-005 and IL7R-006)morpholino SM-ASOs using the Endo-Porter transfection system (GeneTools) as before. FIG. 4A shows splicing analysis of exon 6 intranscripts from the endogenous IL7R gene. Total RNA was isolated fromcells and percentage of exon 6 inclusion was determined by RT-PCR asbefore. FIG. 4B shows the quantification of soluble IL7R (sIL7R)secretion. Secretion of sIL7R was quantified by ELISA in supernatantscollected from cells in panel A. Data are shown as average absorbancefor experimental SM-ASOs normalized to control (ASO-Ctrl). FIG. 4C showsthe quantification of cell surface expression of membrane-bound IL7R(mIL7R). Cell surface expression of mIL7R was determined by flowcytometry with IL7R staining in intact cells. Data are shown as meanfluorescence intensity (MFI) of IL7R staining normalized to control.FIG. 4D shows the ratio of IL7R protein isoforms expression. Ratio ofmIL7R to sIL7R (mIL7R/sIL7R) was determined by dividing values of mIL7Rcell surface expression (FIG. 4C) by sIL7R secretion (FIG. 4B). In allpanels, statistical significance was assessed by two-tailed Student'st-test comparing experimental SM-ASOs versus control (*p<0.05,**p<0.005, ***p<0.0005).

These results demonstrate that SM-ASOs targeting sequences in IL7R exon6 or intron 6 not only induce the desired outcome in exon 6 splicing butmore importantly in the ratio of IL7R protein isoforms (mIL7R/sIL7R).Prominently, IL7R-005 and IL7R-006 decreased sIL7R levels with minimalimpact on mIL7R cell surface expression. The latter is crucial fortreatment of MS and autoimmunity, since inhibition of mIL7R expressionor activity causes immunosuppression. The SM-ASOs IL7R-005 and IL7R-006meet the first efficacy endpoint, which is to restore IL7R proteinisoforms. Importantly, these SM-ASOs are predicted to be a safe and yeteffective therapeutic drug for MS and other autoimmune diseases viamechanisms that prevent immunosuppression.

In addition, these analyses show that IL7R SM-ASOs that decreased exon 6inclusion (IL7R-001 and IL7R-004), effectively increased sIL7R levels,meeting the first efficacy endpoint for a potential cancerimmunotherapy. These SM-ASOs are predicted to enhance the power of theimmune system to fight off and eradicate cancer cells.

FIGS. 5A to 5D show the dose-response modulation of IL7R exon 6 splicingof lead SM-ASOs that reduce sIL7R. HeLa cells stably expressing thefluorescent reporter were transfected with increasing concentrations [0,1, 5, and 10 μM] of control (ASO-Ctrl=0 μM) or experimental (IL7R-005and IL7R-006) morpholino SM-ASOs as before. FIGS. 5A and 5B showanalysis of GFP expression. GFP mean fluorescence intensity (MFI) wasmeasured by flow cytometry as before. FIG. 5A shows representativehistograms of GFP MFI for IL7R-005 at different concentrations, whereasFIG. 5B shows normalized GFP MFI as a function of SM-ASO concentration.FIGS. 5C and 5D show splicing analysis of IL7R exon 6 in transcriptsfrom the reporter (FIG. 5C) or endogenous gene (FIG. 5D). The percentageof exon 6 inclusion in transcripts from the reporter or the endogenousgene was determined as before and is shown as a function of SM-ASOsconcentration. In all panels, statistical significance was assessed bytwo-tailed Student's t-test comparing experimental concentrations versuscontrol (*p<0.05, **p<0.005, ***p<0.0005).

FIGS. 6A to 6D show the dose-response modulation of IL7R exon 6 splicingof lead SM-ASOs that increase sIL7R. HeLa cells stably expressing thefluorescent reporter were transfected with increasing concentrations [0,1, 5, and 10 μM] of control (ASO-Ctrl=0 μM) or experimental (IL7R-001and IL7R-004) morpholino SM-ASOs as before. FIGS. 6A and 6B showanalysis of GFP expression. GFP mean fluorescence intensity (MFI) wasmeasured by flow cytometry as before. FIG. 6A shows representativehistograms of GFP MFI for IL7R-001 at different concentrations, whereasFIG. 6B shows normalized GFP MFI as a function of SM-ASO concentration.FIGS. 6C and 6D show splicing analysis of IL7R exon 6 in transcriptsfrom the reporter (FIG. 6C) or endogenous gene (FIG. 6D). The percentageof exon 6 inclusion in transcripts from the reporter or the endogenousgene was determined as before and is shown as a function of SM-ASOsconcentration. In all panels, statistical significance was assessed bytwo-tailed Student's t-test comparing experimental concentrations versuscontrol (*p<0.05, **p<0.005, ***p<0.0005).

The analyses in FIGS. 5A to 5D and 6A to 6D demonstrate that IL7R exon 6splicing can be fine-tuned in a dose-dependent manner by manipulatingthe dose of SM-ASOs used, and uncovered the minimal effectiveconcentration for IL7R splicing modulation in cell culture. Importantly,these results could be used to extrapolate in vivo dosing inpre-clinical studies in nonhuman primates and clinical trials in humans.This dose-response analysis illustrates an example of thedose-responsiveness of lead SM-ASOs, and does not limit our applicationsto the range of concentrations tested.

FIGS. 7A to 7B show correction by IL7R-005 of the abnormal exclusion ofIL7R exon 6 driven by the MS-associated SNP rs6897932. Hela cells stablyexpressing versions of the GFP-IL7R reporter containing either theprotective ‘T’ allele or the risk ‘C’ allele of the MS-associatedvariant rs6897932 in IL7R exon 6 were transfected with either controlSM-ASO (ASO-Ctrl) or IL7R-005. FIG. 7A shows splicing analysis of IL7Rexon 6 in transcripts from the GFP-IL7R reporter containing thealternative alleles of rs68978932 (C or T). Percentage exon 6 inclusionwas determined by RT-PCR as before. FIG. 7B shows analysis of GFPexpression in cells from FIG. 7A. GFP mean fluorescence intensity (MFI)was measured by flow cytometry as before, and is shown normalized tocells expressing the ‘T’ reporter treated with control SM-ASO. In allpanels, statistical significance was assessed by two-tailed Student'st-test comparing experimental SM-ASOs versus control or as indicated(*p<0.05, **p<0.005, ***p<0.0005).

Previous studies by the present inventors and others found the risk ‘C’allele of the genetic variant rs6897932 (C or T) in IL7R exon 6 toincrease MS risk by enhancing exclusion of exon 6 and sIL7R levels(Gregory et al., 2007; Evsyukova et al., 2013; Hoe et al., 2010;Lundstrom et al., 2013). The analysis above demonstrates that IL7R-005,the lead SM-ASO for treatment of autoimmunity, restores the effects ofthe risk ‘C’ allele of rs6897932, which strongly supports thetherapeutic potential of IL7R-005.

Thus, the present inventors have described compositions and methods forusing antisense oligonucleotides to control alternative splicing of exon6 of the Interleukin 7 receptor (IL7R) RNAs for therapeutic interventionin autoimmunity and cancer. These antisense oligonucleotides controlsplicing of IL7R exon 6 by blocking specific signals embedded in IL7RRNAs. These signals are specific sequences that determine the splicingoutcome of IL7R exon 6. Further, specific sequences in IL7R RNAs to beblocked by antisense oligonucleotides to reduce sIL7R are listed inTable 1, including variations of these sequences, any portion of thesesequences, or any nucleotides flanking these sequences that increaseinclusion of IL7R exon 6, thus decreasing sIL7R secretion. Further,additional signals in IL7R RNAs to be blocked by antisenseoligonucleotides to increase sIL7R are listed in Table 2, includingvariations of these sequences, any portion of these sequences, or anynucleotides flanking these sequences that decrease inclusion of IL7Rexon 6, thus increasing sIL7R secretion. Blocking just a few nucleotidesof these sequences can affect splicing of exon 6 because the actualelement(s) that drives exclusion/inclusion is not the entire targetedsequence but usually a sequence of 4-8 nt within the targeted sequence.For example, an important sequence within the IL7R-005 target sequenceis the last 5 nt UGGUC, thus, an ASO blocking just this 5 nt sequence ora few nt of this sequence might be sufficient to cause the desiredeffect. However, in order to maximize targeting specificity andefficiency for the functional sequence the oligonucleotide is often madeto a longer complementary sequence. Finally, it is well known that it ispossible to replace one or more bases in the antisense oligonucleotideto modify base-pairing while retaining high affinity, selectivity andefficient antisense activity for said target sequences. Depending on thelength of the SM-ASO, non-limiting examples are those having 15, 20, or25 nucleotides, which may have 70, 75, 80, 84, 85, 87, 88, 90, 92, 93,94, 95, or 96, percent identity to any of the SEQ IDs in Tables 1 and 2,or portions thereof, either alone or in combination, fully or partially,or any biologically active permutation of these SEQ IDs for thetreatment of autoimmune diseases, inflammatory diseases or cancer,respectively, or sequences complementary thereto, for use as acomposition and in methods for reducing the expression of soluble IL7Rby enhancing the inclusion of IL7R exon 6 for treatment of autoimmuneand/or inflammatory diseases, or for enhancing the expression of solubleIL7R by reducing the inclusion of IL7R exon 6 for treatment of cancer.For a SM-ASO having 5, 10, 15, 20 or 25 nucleotides the mismatch may be,e.g., 1, 2, 3, 4, 5 or more mismatches.

TABLE 3 Summary of percent identity (% identity) with up to 8 mismatchesfor each of IL7R-005 and IL7R-006. IL7R-005 IL7R-006 IL7R-001 IL7R-004(15 nt) (20 nt) (25 nt) (15 nt) percent percent percent percentmismatches identity identity identity identity 0 100 100 100 100 1 93 9596 93 2 87 90 92 87 3 80 85 88 80 4 73 80 84 73 5 67 75 80 67 6 60 70 7660 7 53 65 72 53 8 47 60 68 47

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps. In embodiments of any of the compositions andmethods provided herein, “comprising” may be replaced with “consistingessentially of” or “consisting of”. As used herein, the phrase“consisting essentially of” requires the specified integer(s) or stepsas well as those that do not materially affect the character or functionof the claimed invention. As used herein, the term “consisting” is usedto indicate the presence of the recited integer (e.g., a feature, anelement, a characteristic, a property, a method/process step or alimitation) or group of integers (e.g., feature(s), element(s),characteristic(s), property(ies), method/process steps or limitation(s))only.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

As used herein, words of approximation such as, “without limitation”,“about”, “substantial” or “substantially” refers to a condition thatwhen so modified is understood to not necessarily be absolute or perfectbut would be considered close enough to those of ordinary skill in theart to warrant designating the condition as being present. The extent towhich the description may vary will depend on how great a change can beinstituted and still have one of ordinary skill in the art recognize themodified feature as still having the required characteristics andcapabilities of the unmodified feature. In general, but subject to thepreceding discussion, a numerical value herein that is modified by aword of approximation such as “about” may vary from the stated value byat least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

To aid the Patent Office, and any readers of any patent issued on thisapplication in interpreting the claims appended hereto, applicants wishto note that they do not intend any of the appended claims to invokeparagraph 6 of 35 U.S.C. § 112, or AIA 35 U.S.C. § 112 paragraph (f), orequivalent, as it exists on the date of filing hereof unless the words“means for” or “step for” are explicitly used in the particular claim.

For each of the claims, each dependent claim can depend both from theindependent claim and from each of the prior dependent claims for eachand every claim so long as the prior claim provides a proper antecedentbasis for a claim term or element.

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What is claimed is:
 1. A composition comprising an oligonucleotide thatis a splice-modulating antisense oligonucleotide (SM-ASO) thatspecifically binds to a sequence of the Interleukin-7 receptor (IL7R)pre-mRNA that influences splicing of exon 6, wherein the SM-ASOincreases inclusion of exon 6 in IL7R pre-mRNAs and decreases expressionof the soluble isoform of IL7R (sIL7R).
 2. The composition of claim 1,wherein the SM-ASO specifically binds to a sequence in IL7R pre-mRNA inat least one of the group consisting of an exonic splicing silencer(ESS) and/or an intronic splicing silencer (ISS), thereby enhancinginclusion of exon 6 in IL7R pre-mRNAs, and reducing expression of sIL7R.3. The composition of claim 1, wherein at least one or morenucleotide(s) in the SM-ASO contains a non-naturally occurringmodification comprising modifications or substitutions of: (1) theribose or other sugar units, (2) bases, or (3) the backbone, selectedfrom: one or more phosphorothioate, phosphorodithioate, phosphodiester,methyl phosphonate, phosphoramidate, methylphosphonate, phosphotriester,phosphoroaridate, morpholino, amidate carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, alkylsilyl substitutions, partially or completelymodified backbones, such as fully modified sugar phosphate backbone, alocked nucleic acid backbone, a peptidic backbone, a phosphotriesterbackbone, a phosphoramidate backbone, a siloxane backbone, acarboxymethylester backbone, an acetamidate backbone, a carbamatebackbone, a thioether backbone, a bridged methylene phosphonatebackbone, a phosphorothioate backbone, a methylphosphonate backbone, analkylphosphonate backbone, a phosphate ester backbone, analkylphosphonothioate backbone, a phosphorodithioate backbone, acarbonate backbone, a phosphate triester backbone, a carboxymethyl esterbackbone, a methylphosphorothioate backbone, a phosphorodithioatebackbone, a backbone having p-ethoxy linkages, sugar modifications suchas 2′-O-methyl (2′-O-methylnucleotides), 2′-O-methyloxyethoxy(2′-O-MOE), a 2′-O-alkyl modified sugar moiety, or a bicyclic sugarmoiety, nucleotide mimetics, peptide nucleic acids (PNA), morpholinonucleic acids, cyclohexenyl nucleic acids, anhydrohexitol nucleic acids,glycol nucleic acid, threose nucleic acid, and locked nucleic acids(LNA), and any combinations of two or more of any of the foregoing. 4.The composition of claim 1, wherein at least one or more nucleotide(s)in the oligonucleotide contains a non-naturally occurring modification.5. The composition of claim 1, wherein the composition further comprisesa pharmaceutically acceptable excipient, salts, or carrier.
 6. Thecomposition of claim 1, wherein the composition is adapted foradministration to treat an autoimmune disorder selected from at leastone of the following: multiple sclerosis, type I diabetes, rheumatoidarthritis, systemic lupus erythematosus, atopic dermatitis, ankylosingspondylitis, primary biliary cirrhosis, or inflammatory bowel syndromessuch as ulcerative colitis and Crohn's disease, or any other conditionwhere sIL7R is elevated.
 7. The composition of claim 6, wherein thecomposition avoids immunosuppression.
 8. The composition of claim 1,wherein the oligonucleotide is in an expression vector.
 9. Thecomposition of claim 1, wherein the oligonucleotide targets any of SEQID NOS: 1 to 13, whether fully or partially.
 10. A composition forincreasing inclusion of exon 6 in an Interleukin-7 receptor (IL7R)pre-mRNA comprising a splice modulating antisense oligonucleotide(SM-ASO) that specifically binds to a sequence in the Interleukin-7receptor (IL7R) pre-mRNAs and increases inclusion of exon 6 in IL7Rpre-mRNAs and decreases expression of the soluble isoform of IL7R(sIL7R).
 11. The composition of claim 10, wherein the SM-ASOspecifically binds to a sequence in IL7R pre-mRNA in at least one of thegroup consisting of an exonic splicing silencer (ESS) and/or an intronicsplicing silencer (ISS), thereby enhancing inclusion of exon 6 in IL7Rpre-mRNAs, and reducing expression of sIL7R.
 12. The composition ofclaim 10, further comprising a combination therapy of the SM-ASO withone or more active agents effective for treating autoimmune orautoimmune diseases selected from mitoxatrone, interferon beta-1a, PEG-interferon beta-1a, azathioprine, fingolimod, natalizumab,methylprednisolone, or ocrelizumab.
 13. The composition of claim 10,wherein the SM-ASO is expressed by a vector.
 14. The composition ofclaim 13, wherein the vector is an expression vector.
 15. Thecomposition of claim 13, wherein the vector is a viral vector or aplasmid.
 16. The composition of claim 13, wherein the vector is used ingene therapy to treat a patient.
 17. The composition of claim 16,wherein the SM-ASO targets SEQ ID NOS: 1 to 13 or portions thereof.